Glioblastoma (GBM) represents the most aggressive and lethal form of primary brain cancer. We now know that GBM contains small subsets of cells that display tumor-propagating stem-like phenotypes (GBM stem cells, GSCs) that act as critical determinants of GBM resistance to current treatments and tumor recurrence. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor- propagating cell subsets is critical to improving GBM therapy and patient outcomes. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and role in the tumor propagating GSC phenotype. This proposal builds on our published and preliminary findings that oncogenic reprogramming transcription factors induce tumor propagating GBM stem cells by regulating miRNA networks that target determinants of DNA methylation. Our new preliminary data implicate a previously unrecognized Sox2:miR-10b-5p:Tet2 axis by which Sox2 induces onco-miR10b-5p that represses the Tet2 demethylase in GBM cells. This axis decreases the conversion of 5-methylcytosine to 5- hydroxymethylcytosine (5hmC), the reaction catalyzed by TET (Ten Eleven Translocation) proteins and a key intermediate step in DNA de-methylation. The potential significance of this axis on oncogenic transcriptome generation is supported by clinical data showing that miR-10b-5p expression is substantially elevated in GBM, that low Tet2 and low 5-hmc expression correlate with poor prognosis in GBM patients, and that Tet2 knock- down accelerates the invasive growth of GBM xenografts. This proposal will test the hypothesis that Sox2 drives tumor-propagating GSCs by activating onco-miR-10b-5p that represses Tet2 resulting in an oncogenic DNA methylome and transcriptome. These goals will be achieved through the following specific aims: (i) Determine how the Sox2:miR-10b:Tet2 axis modifies the DNA methylation landscape of GSCs; (ii) Determine how enzymatic and non-enzymatic functions of Tet2 regulate GSCs; (iii) Determine the potential to treat GBM in vivo by inhibiting Tet2 down-regulation by miR-10b-5p. Successful execution of the proposed research plan will fill critical gaps in our understanding of epigenetic molecular events by which reprogramming transcription factors induce tumor propagating cells in GBM through transcriptional silencing, and establish the potential to treat GBM by targeting this novel axis that alters the tumor transcriptome via Tet2 inhibition.